744 Blood feeding in juvenile Paragnathia formica (: Gnathiidae): biochemical characterization of trypsin inhibitors, detection of anticoagulants, and molecular identification of fish hosts

B. M. MANSHIP, A. J. WALKER*, L. A. JONES and A. J. DAVIES School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK

(Received 21 July 2011; revised 7 October 2011; accepted 14 November 2011; first published online 6 February 2012)

SUMMARY

The 3 post-marsupial juvenile stages of the gnathiid isopod, Paragnathia formica, are haematophagous ectoparasites of fishes that may, in heavy infestations, cause host mortality. Protein digestion in fed stage 3 juveniles is accomplished by cysteine proteinases, but what bioactive compounds attenuate host haemostatic, inflammatory and immunological responses during feeding is unknown. Trypsin inhibitory activity and anticoagulant activity were detected in crude extracts of unfed P. formica stage 1 juveniles; fractionation of stage 1 crude extracts by ion exchange chromatography resulted in 3 preparations each displaying these bioactivities. Further characterization revealed anti-thrombin activity in 2 of these preparations, whilst the third displayed the strongest anticoagulant activity that targeted a factor of the intrinsic coagulation pathway. Three trypsin inhibitors (18 kDa, 21 kDa, and 22 kDa) were also detected using reverse zymography. In parallel, homogenates of fed stage 2 and 3 juveniles were used to identify their fish hosts by amplifying the 16S mitochondrial rDNA and 18S genomic rDNA vertebrate gene regions. Blood from at least 4 fish families had been ingested by separate individuals during feeding. This study demonstrates that trypsin inhibitors and anticoagulants are present in P. formica juveniles which could suppress host haemostatic, inflammatory and immunological responses during feeding, and that juveniles are not host specific.

Key words: Paragnathia formica, fish ectoparasite, blood feeding, host-parasite interactions, serine protease inhibitor, anticoagulants.

INTRODUCTION The present study focuses on the gnathiid Paragnathia formica (Hesse, 1864), which inhabits Gnathiid isopods (Crustacea; Isopoda; Gnathiidae) mud microcliffs in estuaries across Western Europe, have biphasic life cycles with free-living adults North Africa and the Mediterranean Basin (Monod, and parasitic, post-marsupial juvenile stages feeding 1926; Upton, 1987; Cadée et al. 2001; Silva et al. on fishes. Although approximately 187 gnathiid 2006; Kirkim et al. 2008). Adult and juvenile stages species have been described world-wide, inhabiting can be collected readily from galleries in the mud estuaries, intertidal zones, coral reefs, and the deep banks, making this species an excellent gnathiid sea, the impact of blood feeding by their juvenile model for study. Mature females, in contact with stages is difficult to assess since they mostly target water, release unfed zuphea 1 juveniles. These wild fishes (see Smit and Davies, 2004; Jones et al. attach to host fish epithelia and, using piercing 2007; Tanaka, 2007; Manship, 2009; Ferreira et al. mouthparts, penetrate host tissues, feeding on 2009, 2010). The parasite-host association can cause whole blood, plasma and possibly mucus, to become host stress, lesions, secondary infections, anaemia, inflated praniza 1 stages (Smit and Davies, 2004). As and mortalities in captive environments (see Hayes in other gnathiids, three zuphea (unfed) and three et al. 2011). Like some terrestrial haematophagous praniza stages (fed), as well as adults, occur in the life , gnathiids have also been implicated in cycle of P. formica. the transmission of blood dwelling apicomplexans In fed praniza forms of P. formica, the bloodmeal between fishes, though the route of transmission undergoes initial breakdown in the anterior hindgut remains uncertain (see Davies and Smit, 2001;Smit and protein digestion by cysteine-like proteinases in and Davies, 2004;Smitet al. 2006). paired digestive glands (Manship et al. 2008). These processes provide amino acids that enable growth and moulting of praniza stages 1, 2, and 3 to subsequent * Corresponding author: Tel: +44 208 547 2466. Fax: zuphea 2, and 3 stages, or to adults; they also sustain +44 208 547 7562. E-mail: [email protected] survival and reproduction of non-feeding adults, and

Parasitology (2012), 139, 744–754. © Cambridge University Press 2012 doi:10.1017/S0031182011002320 Blood feeding in Paragnathia formica 745 support embryological development (see Smit and MATERIALS AND METHODS Davies, 2004; Manship et al. 2011). In some respects Specimen sampling, maintenance and storage the feeding behaviour of gnathiids resembles that of ticks (Ribeiro et al. 1985); P. formica for example can Paragnathia formica praniza 2 and 3 juveniles (n=7) remain attached to the host for hours to days (Stoll, and gravid females (n=*600) were collected from 1962). This association suggests that gnathiids, like salt marsh banks of the type described by Manship ticks, must evade host defence responses, including et al.(2008, 2011) at Wells-next-the-Sea, Norfolk haemostasis, which limits blood loss by vasoconstric- (Ordnance Survey grid reference, TF 925 438) (see tion, platelet aggregation and blood coagulation Tinsley and Reilly, 2002) during April and May, (Ribeiro and Francischetti, 2003). Blood coagulation and from the Dovey Estuary, Wales (Ordnance in fishes resembles that in higher vertebrates, with an Survey grid reference, SN 674 973) during July and extrinsic pathway stimulated by a tissue factor mostly September. Live praniza juveniles were rinsed in found in skin, and thrombocytes replacing mam- distilled water, fixed in ice cold 70% ethanol and malian platelets in the intrinsic pathway (Tavares- stored at 4 °C. Live females were maintained on Dia and Oliveira, 2009; Doolittle, 2011). damp tissue paper (in the dark) at 16 °C in an While haematophagous aquatic ectoparasites of incubator, and were transferred to sea water, once fishes likely release anti-haemostatic compounds into mature, for release of motile zuphea 1 juveniles. their hosts during feeding, research on such com- Spent females were removed from the sea water, pounds in these is scarce, limited largely to a whilst the unfed juveniles were further maintained in report by Romestand and Trilles (1976a,b) demon- sea water for 1 week (Tinsley and Reilly, 2002). After strating anti-thrombin activity towards fish blood rinsing in distilled water, zuphea 1 juveniles 3 in the salivary glands of adult cymothoid isopods (approximately 1 g in total, or *53×10 individuals) Meinertia oestroides (Risso, 1826) and Anilocra were collected on absorbent filter paper, snap frozen physodes (Linnaeus, 1758) (Isopoda: Cymothoidae). in liquid nitrogen and stored at −80 °C. Because zuphea 1 stages of P. formica have well developed salivary glands, even before hatching Biochemical analyses and semi-purification of from females (Manship et al. 2011), we hypothesized trypsin inhibitors and anticoagulants from unfed that these glands might produce pharmacological zuphea 1 stages compounds such as anticoagulants to facilitate blood feeding by P. formica. Here, we demonstrate for Zuphea 1 forms were selected since only the newly the first time the partial purification of trypsin hatched juvenile stages of P. formica could be inhibitors, and anticoagulant activities partly target- harvested in sufficient numbers to permit bio- ing thrombin, in recently-hatched P. formica zuphea chemical analyses of trypsin inhibitors and anti- 1 juveniles. coagulants. Approximately 1 g of zuphea 1 stages As in ticks (Pichon et al. 2003), gnathiids parasitize stored at −80 °C (above) were homogenized in a a variety of hosts, sometimes demonstrating micro- sterile Potter homogenizer on ice in 5 ml of 50 mM predatory behaviour, though little is known of their Tris-HCl buffer, pH 8·5, containing 20 mM NaCl host-selectivity. Such knowledge could be vital for (Morris and Sakanari, 1994; Azzolini et al. 2003), assessing the impact of their feeding behaviour in centrifuged at 14000 g for 5 min at 4 °C, and the various ecosystems, for instance on the Great Barrier crude extract (CE) protein concentrations measured Reef (GBR), Australia (Nagel and Lougheed, 2006; using the Bio-Rad DC microassay according to the Jones et al. 2007), or on the transmission of manufacturer’s instructions. apicomplexan parasites (Smit and Davies, 2004; The inhibitory effect of the zuphea 1 CE (approxi- Smit et al. 2006). Host specificity in some gnathiids mate protein concentration =10–14·5 μg/μl) on the of the genus Leach, 1814 has been partially proteolytic activity of trypsin towards the fluorogenic addressed on the GBR, by sequencing specific serine protease substrate N-carbobenzoxy-Gly-Pro- vertebrate 12S and 16S rDNA mitochondrial gene Arg-4-methoxy-2-naphthylamine (Z-gly-pro-arg- regions from fed (praniza) gnathiids (Nagel and MNA; Bachem) was then determined according Lougheed, 2006; Jones et al. 2007). To date, several to Morris and Sakanari (1994) and Azzolini et al. P. formica host fishes have been recorded from (2003). Varying amounts of CE (0 μg (control), the wild (Monod, 1926; Menezes, 1984; Kirkim 0·2 μg, 1 μg, 2 μg, 5 μg10μg, 20 μg, 50 μg, and et al. 2008), and in laboratory feeding experiments 100 μg) were pre-incubated for 15 min at room (Monod, 1926), which suggest that this gnathiid is temperature with 1 nM (0·006 U) trypsin from relatively host non-specific. Here, we verify that bovine pancreas (Calbiochem) in 50 mM Tris-HCl several host fishes can be identified from the blood- buffer, pH 7·5, within individual wells of a microtitre meal content of praniza juveniles collected from two plate; 10 μl of Z-gly-pro-arg-MNA (0·1 mM final sites in the UK, using PCR amplification targeting concentration in 100 μl total reaction volume) were conserved gene sequences, namely 16S rDNA and then added and the released MNA (excitation 18S rDNA gene regions. 355 nm, emission 420 nm) measured over 2 h at B. M. Manship and others 746

25 °C using a Fluostar Optima reader (BMG (1983) and Andreotti et al.(2002). The gel was Labtech). The inhibitory capacity of the CE on the electrophoresed at 4 °C at 140 V, and subsequently hydrolysis of 0·1 mM Z-gly-pro-arg-MNA (Bachem) washed in 0·1 M Tris-HCl, pH 8·0, containing 2·5% by thrombin was determined by pre-incubating (v/v) Triton X-100 for 1 h. The gel was then rinsed in 50 nM (0·009 U) bovine thrombin (Calbiochem) distilled water and incubated at 37 °C for 90 min in with 20 μg CE for 15 min at room temperature in 25 ml of 0·1 M Tris-HCl buffer, pH 8·0, containing 50 mM Tris-HCl buffer, pH 7·5. The reaction was 87 μM (125 000 U) bovine trypsin. The gel was then initiated by adding the substrate (0·1 mM final rinsed in distilled water and stained with Coomassie concentration in 100 μl total reaction volume) and brilliant blue to reveal bands corresponding to the inhibition measured as for the trypsin inhibitor molecular weight(s) of the trypsin inhibitors. assays. Finally, the effects of the semi-purified prepa- Effects of the zuphea 1 CE on the recalcification rations on human plasma and fibrinogen clotting time and fibrinogen clotting time of human plasma times were determined as described above. Briefly, were assayed according to Ricci et al.(2007). For 2·7 μg, 7·6 μg and 8·1 μg of P1, P2, and P3, res- recalcification time, 25 μl citrated human plasma pectively, were assayed for their effects on recalcifica- (Sigma) were incubated at room temperature in tion time (at 50% clot formation) and fibrinogen 50 mM Tris-HCl buffer (final volume 70 μl), clotting time (at 50% clot formation), against buffer in pH 7·5, containing 0 μg (control), 14·5 μg, 29·1 μg, control wells. In the case of the recalcification time, or 72·5 μg zuphea 1 CE. After 5 min, coagulation was 2·7 μg P1 (possessing the greatest anticoagulant initiated by adding 5 μl 150 mM CaCl2, and clot activity) were boiled for 10 min prior to conducting formation was continuously measured in microtitre the assay to verify whether the anticoagulant(s) could plates at 25 °C for 30 min at 620 nm with a Fluostar be heat denatured, and thus, be proteinaceous. Optima reader. For the fibrinogen clotting assay, All enzyme/coagulation assays were carried out 6·5 μM fibrinogen from human plasma (Calbiochem) in triplicate, and one-way analyses of variance was incubated at room temperature in 50 mM (ANOVA) and post-hoc multiple comparison tests Tris-HCl buffer (total incubation volume of 90 μl), (Least Significant Difference, LSD) were done on pH 7·5, containing 0 μg (control), 14·5 μg or 72·5 μg data obtained. CE. After 5 min, 10 μl of a 0·54 μM (2·2 U) solution of thrombin from human plasma (Calbiochem) were added to trigger clot formation, which was measured Molecular typing of vertebrate DNA from fed at 620 nm, as described for the recalcification time; praniza juveniles the final absorbances measured were subtracted from values obtained at 0 min. Zuphea 1, and subsequent zuphea 2 and 3 stages of The zuphea 1 CE was then applied to a Nap-25 P. formica feed on fishes, becoming pranizae 1, 2 and column (GE Healthcare) at 4 °C according to the 3 respectively. Thus, to investigate the source of host manufacturer’s instructions. Fractions collected from blood in the juvenile gnathiid digestive tract, larger the Nap-25 column displaying inhibitory activity fed praniza 2 (2–2·5 mm) and 3 (3–3·5 mm) stages were pooled, and concentrated to 1 ml using a were employed. A set of universal (vertebrate and Vivapore® concentrator (Vivascience). In an attempt invertebrate) primers targeting the mitochondrial to purify the bioactive compound(s) from the zuphea large subunit ribosomal DNA (16Sar and 16Sbr) 1 homogenate, the concentrated sample was applied (Simon, 1991), as well as the eukaryote conserved to a 1 ml Sepharose anion-exchange chromatography 18SA (Blakenship and Yayanos, 2005) primer and a column (HiTrap Q) (GE Healthcare) at 4 °C pre- reverse 18S primer modified from Dreyer and Wägele equilibrated with Tris-HCl wash buffer (Tanaka (2001) to anneal the genomic small subunit ribosomal et al. 1999). This resin was used since its working DNA of fishes, were used. DNA was extracted from pH range is optimal for the stability of trypsin blood-filled, individual, praniza juveniles using a and trypsin inhibitors. After washing with 50 mM protocol modified from Sicard et al.(2001) and Tris-HCl, pH 8·5, at 1 ml/min, bound proteins were Jones et al.(2007). Briefly, specimens were rinsed in eluted with 50 mM Tris-HCl, pH 8·5, containing a distilled water and placed individually in sterile NaCl gradient of 0–0·5 M (in 10 mM increments), microfuge tubes containing 200 μl DNA extraction and 1 ml fractions collected on ice. Three major peaks buffer (1 M Tris-HCl, 5 M NaCl, 0·5 M EDTA, (P1, P2, and P3) of trypsin inhibitory activity were 0·2% (v/v) β-mercaptoethanol, and 5% (v/v) CTAB). obtained and concentrated. Specimens were then homogenized using sterile To determine the size of the trypsin inhibitors plastic pestles (Anachem) and incubated for 1 h at isolated in the fraction displaying the strongest 55 °C, prior to the addition of 200 μl of chloroform. trypsin inhibitory activity, 6·5 μg P1 were loaded After centrifugation, the aqueous phase containing onto 15% SDS-PAGE gels, either containing 0·1% genomic DNA was added to 200 μl of ice-cold (w/v) gelatin or not, and those with gelatin processed isopropanol to precipitate the DNA. After rinsing for reverse zymography according to Hanspal et al. in 70% ethanol, precipitated nuclear material was Blood feeding in Paragnathia formica 747

(A)

Fig. 1. Effect of varying amounts of Paragnathia formica zuphea 1 crude extract (CE) on the hydrolysis of 0·1 mM (B) Z-gly-pro-arg-MNA by 1 nM trypsin. Bars represent mean values of 3 replicates (± S.D.), from both control (&) and CE (□) assays. ** P<0·01 and *** P<0·001 when compared to control values. re-pelleted and re-suspended in 15 μl of diethylpyr- ocarbonate (DEPC)-treated water. Extracted DNA (5 μl per 50 μl assay) was amplified using the BD TITANIUM™ Taq (BD Biosciences), and primers noted above, following the cycling programme used by Jones et al.(2007). The *600– 650 bp 16S rDNA and *300–350 bp 18S rDNA bands were extracted from the agarose gel using a Fig. 2. Anticoagulant properties of Paragnathia formica QIAquick gel extraction kit (Qiagen), and sequenced zuphea 1 crude extract (CE). (A) Recalcification time at the Natural History Museum (South Kensington, of human plasma in the absence (♦) or presence of London). 14·5 μg(□), 29·1 μg (*), or 72·5 μg (×) of zuphea 1 CE; Sequences were entered into the Basic Local (B) Fibrinogen clotting time of human fibrinogen in the ♦ μ □ μ Alignment Search Tool (BLAST) (http://www.ncbi. absence ( ) or presence of 14·5 g( ), or 72·5 g (×) nlm.nih.gov/BLAST/) in order to obtain matching of zuphea 1 CE. Data represent the mean values of 3 replicates. sequences (McGinnis et al. 2004). The amplified sequences were subsequently aligned against the closest matching sequences using CLUSTAL W approximately 30% (P<0·01) (data not shown). (http://clustalw.genome.jp/)(Thompsonet al. 1994), Owing to the higher level of trypsin inhibition, and whenever sequence manipulation was required compared to thrombin inhibition by the zuphea the molecular toolkit program (http://www.vivo. 1 CE, subsequent experiments focused primarily colostate-edu/molkit/manip/) was employed. FishBase on trypsin inhibition. A dose-dependent inhibition (http://www.fishbase.org/) was scrutinised to verify of trypsin activity by the CE was observed; Z-gly- whether matching fish species were compatible hosts pro-arg-MNA hydrolysis by trypsin was reduced by μ μ for gnathiids inhabiting the Atlantic coast and North 50% with 2 gCE(P<0·001), 91·6% with 50 gCE μ Sea coast. (P<0·001), and over 95% with 100 gCE(P<0·001); in contrast no inhibition was observed with 0·2 μgCE (Fig. 1). RESULTS To assess the effect of the zuphea 1 CE on coagulation of human plasma, a coagulation assay Characterization of trypsin inhibitors and estimating the recalcification time of human plasma anticoagulants from zuphea 1 extracts was conducted. The mean time to reach 50% Crude zuphea 1 extracts were assayed for the clot formation in the control sample was 15 min presence of trypsin or thrombin inhibitors using the 10 s. Pre-incubation with 14·5 μg or 29·1 μg zuphea 1 serine protease substrate Z-gly-pro-arg-MNA. The CE prolonged 50% clot formation by 3 min 20 s proteolytic activity of trypsin towards this substrate (±52 s) and 6 min 10 s (±2 min 30 s), respectively, was inhibited significantly by over 80% (P<0·001) representing mean increases of 22% and 40·7% after pre-incubation in buffer containing 20 μg (Fig. 2A). Moreover, pre-incubation with, 72·5 μg zuphea 1 CE, whereas the proteolytic activity of CE inhibited clot formation entirely over 30 min thrombin towards the same substrate diminished by (Fig. 2A). To substantiate the inhibitory activity of B. M. Manship and others 748

(A) (B)

Fig. 3. Protein concentration (bars) and trypsin inhibitory activity (♦)ofParagnathia formica zuphea 1 fractions collected from a HiTrap Q ion-exchange Fig. 4. (A) Coomassie-stained SDS-PAGE gel showing μ chromatography column. Approximately 15 mg of zuphea the low molecular weight ladder (lane 1), and 6·5 g 1 protein recovered from the Nap-25 gel filtration column Paragnathia formica zuphea 1 peak 1 collected from the were passed through the HiTrap Q column at a flow rate HiTrap Q ion-exchange chromatography column (lane 2). of 1 ml/min. Proteins captured by the column were eluted (B) Coomassie stained reverse zymography gel showing μ after 13 min by applying increasing NaCl concentrations the trypsin inhibitory activity of 6·5 g peak 1 (lane 1) (0 to 0·5 M in 0·01 M increments every 30 s). The and the low molecular weight ladder, in particular the * fractions were recovered, protein concentration measured inhibitory activity of SBTI at 20 kDa (lane 2). Trypsin and 10 μl of each fraction incubated with 1 nM trypsin for inhibitors were detected using 15% polyacrylamide 15 min prior to the addition of 0·1 mM Z-gly-pro-arg- resolving gels containing 0·1% gelatin. After MNA to determine trypsin inhibitory activity. Three electrophoresis at 4 °C, the gels were incubated for 90 min μ inhibitory peaks were detected (delineated by the dotted in 87 M trypsin at 37 °C. Undigested areas by the ff vertical lines), each comprising 5 fractions, between trypsin produced di use inhibition zones, similar to the * μ 0·15–0·23 M NaCl (P1), 0·24–0·33 M NaCl (P2) and 20 kDa band produced by approximately 2·2 g SBTI 0·34–0·43 M NaCl (P3). contained in the low molecular weight ladder.

obtained using 2·7 μg of the semi-purified prep- zuphea 1 CE towards thrombin seen in the fluori- aration P1 to those observed using 50 μg zuphea 1 CE metric assays, and to characterize further the anti- (Fig. 1), indicating that the partial purification of coagulant effect, fibrinogen was pre-incubated with the trypsin inhibitor(s) from the crude extract was zuphea 1 CE prior to the addition of thrombin. After successful. 30 min, 72·5 μg CE prolonged 50% fibrinogen clot Following the partial purification of trypsin formation by 1 min 40 s (± 1 min) compared to the inhibitor(s) in the zuphea 1 CE, reverse zymography uninhibited control wells that possessed a mean 50% was done using protein from the most active peak clotting time of 8 min 40 s; no delay was seen with (P1) to determine the sizes of the trypsin inhibitor(s) 14·5 μg CE per assay (Fig. 2B). present. Soybean trypsin inhibitor (SBTI) (20 kDa) Zuphea 1 protein (30 mg) was desalted using in the molecular weight markers served as a positive a Nap-25 column and subsequently applied to a control. The large diffuse band obtained (Fig. 4B, HiTrap Q column. A total of 3–4 mg of zuphea 1 lane 2) corresponded to the location of SBTI protein was collected in 38 fractions (Fig. 3), and 3 (Fig. 4A, lane 1) after trypsin digestion of the gelatin major protein peaks corresponding to 3 separate within the gel. In the case of the semi-purified zuphea trypsin inhibitory peaks were eluted off the HiTrap Q 1 P1, 3 smaller diffuse bands were obtained with column between 0·15–0·23 M NaCl (P1), 0·24–0·33 M apparent molecular weights of *18 kDa, *21 kDa NaCl (P2), and 0·34–0·43 M NaCl (P3) (Fig. 3). and *22 kDa (Fig. 4B, lane 1), which did not seem to The fractions within each of these peaks of trypsin correspond to any of the 6 major bands seen on the inhibition were then pooled. Preparations from peaks SDS-PAGE gel (Fig. 4A, lane 2). This suggests 2 and 3 (P2 and P3) had greater protein concen- that although present in low concentrations, the trations 0·76 μg/μl and 0·81 μg/μl, compared to that inhibitors have potent inhibitory activity. of P1 (0·27 μg/μl); however, they displayed lower The anticoagulant properties of the semi-purified trypsin inhibitory activities. Indeed, 2 μl P2 (1·52 μg fractions were assayed alongside their trypsin inhi- zuphea 1 protein) and P3 (1·62 μg zuphea 1 protein) bitory activities. Of the 3 preparations obtained, inhibited trypsin hydrolysis by 28·2% ±6·4% preparation P1 possessed the greatest anticoagulant (P<0·05) and 44·1%±2·5% (P<0·05), respectively, activity, with 2·7 μg zuphea 1 protein delaying whereas 5 μl (1·35 μg zuphea 1 protein) P1 reduced recalcification time of human plasma (mean time to trypsin hydrolysis by 84·25% ±0·46 (P<0·001) (data 50% clot formation of 15 min 10 s) by 6 min 40 s not shown). Similar levels of trypsin inhibition were (± 3 min 30 s), representing a 44% (P<0·05) increase Blood feeding in Paragnathia formica 749

(A) and P3, and that the predominant anticoagulant(s) contained in the CE were present in P1. Finally, the anticoagulant activity observed in P1 was heat denatured (Fig. 5A) and this no longer inhibited the recalcification time of human plasma, suggesting that the anticoagulant(s) are proteinaceous.

Molecular identification of fish hosts from praniza bloodmeals Fish DNA sequences amplified best with the mitochondrial 16S rDNA primers from P. formica praniza 3 juveniles collected at Wells-next-the-Sea, revealing 3 host families, including the Anguillidae, (B) Clupeidae, and Pleuronectidae (Table 1). Further- more, the 16S rDNA gene region identified hosts successfully to genus and species level, with over 99% homology to the European eel, Anguilla anguilla (Linnaeus, 1758), 95–98% homology to the Atlantic herring, Clupea harengus Linnaeus, 1758, and 98–99% homology to the European flounder, Platichthys flesus (Linnaeus, 1758), all fish species native to North Sea shores. Two host families were identified from praniza 2 and 3 specimens from the Dovey Estuary using the 18S rDNA primers (Table 1). Two pranizae had fed on hosts belonging to the Pleuronectidae, the closest matching fish (with over 97% and 99% homology) Fig. 5. (A) Effect of Paragnathia formica zuphea 1 being the stone flounder, Kareius bicoloratus semi-purified proteins on the recalcification time of (Basilewsky, 1855), a Pacific Ocean fish (Table 1). human plasma after pre-incubation of plasma with 0 μg However, the European plaice, Pleuronectes platessa zuphea 1 protein from semi-purified peaks (♦), 2·7 μgP1 Linnaeus, 1758, has a shorter 18S rDNA sequence ▲ μ μ ○ μ ( ), 7·6 g P2 (+), or 8·1 gP3( ), as well as 2·7 g than the stone flounder, but shares similar homo- zuphea 1 peak 1 previously boiled for 10 min at 100 °C logies (99% and 97%, respectively) with the 2 (Δ). (B) Effect of P. formica zuphea 1 semi-purified sequences obtained from the gnathiid gut contents proteins on the fibrinogen clotting time of human fibrinogen after pre-incubation with 0 μg zuphea 1 protein (Table 1) and is a much more likely host in this from semi-purified peaks (♦), 2·7 μgP1(▲), 7·6 μgP3 instance. In a similar manner, a third praniza (+), or 8·1 μgP3(○). Data represent the mean values of collected from the Dovey Estuary had apparently 3 replicates. fed upon a member of the Scombridae, and in this case the closest match was the bullet tuna, Auxis rochei (Risso, 1810) (Table 1). Since this tuna occurs (Fig. 5A). In contrast, 8·1 μg P3 delayed recalcifica- on European Atlantic coasts, but does not inhabit tion time by 2 min 50 s (±1 min) or 18·7%, and 7·6 μg estuaries, it appears more likely that the gnathiid had P2 did not delay 50% clot formation compared to fed on another scombrid. Thus, it seems that the 18 s control samples (Fig. 5A). However, both P2 and P3 rDNA gene region selected was too conserved to preparations displayed anti-thrombin activities in identify hosts reliably to genus and species level, fibrinogen clotting assays, since the duration to 50% and the host species listed as the closest matching clot formation was delayed by 4 min (± 2 min 10 s) or organisms in the BLAST results were a consequence 46·2%, and by 4 min 10 s (± 4 min 20 s) or 48·1%, of better sequence coverage rather than sequence respectively compared to the mean control clot homology. formation time (8 min 40 s) (Fig. 5B). Only a slight increase 15·4% (±5·8%) in delay in fibrinogen clotting time could be attributed to P1, and although the DISCUSSION protein concentration in this preparation was lower We report for the first time, partial purification than in P2 and P3, inhibitory activity towards by anion-exchange chromatography of different thrombin in this preparation again appeared to follow bioactive compounds from homogenates of newly that observed in the CE (Figs 2B and 5B). This is hatched P. formica zuphea 1 juveniles which could supportive of zuphea 1 anti-thrombin compound(s) attenuate host haemostatic, inflammatory and immu- being present in small amounts and primarily in P2 nological responses. Preliminary trypsin/thrombin B. M. Manship and others 750

Table 1. Identification of vertebrate fish host from the bloodmeal content of praniza 2 and praniza 3 juveniles of Paragnathia formica collected from Wells-next-the-Sea (see Tinsley and Reilly, 2002) and the Dovey Estuary (see Manship et al. 2008)

Bloodmeal origin Targeted genes Host Family Homology Closest matching organism

Wells-next-the-Sea 16S rDNA Anguillidae 99% Anguilla anguillaa Pleuronectidae 99% Platichthys flesusa Clupeidae 98% Clupea harengusa Pleuronectidae 98% Platichthys flesusa Dovey Estuary 18S rDNA Pleuronectidae 99% Kareius bicoloratusb Scombridae 97% Auxis rocheib Pleuronectidae 97% Kareius bicoloratusb a Likely host species; b unlikely hosts=possible congener species. inhibition assays and coagulation assays with zuphea available (1 g total represented *53×103 zuphea 1 1 CE, revealed trypsin and thrombin inhibitors and juveniles) restricted further characterization of the anticoagulants. The anticoagulants delayed the re- anticoagulant(s) found, and the specific factor(s) calcification time of human plasma, an indicator of in the coagulation cascade targeted by these com- blood coagulation via the intrinsic pathway (Abebe pounds. In terrestrial blood-feeding arthropods, et al. 1996; see Tavares-Dia and Oliveira, 2009), and anticoagulant compounds identified so far predomi- fibrinogen clotting time, a test estimating thrombin nantly target common factors to both intrinsic and catalysis of fibrinogen into fibrin (Ricci et al. 2007; extrinsic pathways, such as factor Xa or thrombin (see see Tavares-Dia and Oliveira, 2009). Stark and James, 1996; Valenzuela, 2002; Koh and Following fractionation by ion-exchange chrom- Kini, 2009), and since thrombin activity was not atography, 3 different bioactive peaks were isolated reduced by the bioactive compounds contained in P1, (P1, P2, P3). The first preparation (P1), displayed the these may inhibit factor Xa activity. strongest anti-trypsin activity, and reverse zymogra- Finally, the semi-purified preparations P2 and P3 phy revealed 3 trypsin inhibitors in this fraction with reduced fibrinogen-clotting time, and may contain apparent molecular weights *18 kDa, *21 kDa the thrombin inhibitor(s) detected in the CE using a and *22 kDa. These trypsin inhibitors might target fluorogenic substrate Z-gly-pro-arg-MNA. Although serine protease factors of the coagulation cascade not further characterized, it appears that both (Watanabe et al. 2010) or serine proteases involved in fractions contain different bioactive compound(s), other physiological processes (see Rimphanitchayakit eluted off the anion-exchange chromatography col- and Tassaanakajon, 2010) in this gnathiid isopod. umn at differing NaCl concentrations, which prevent Indeed, serine protease inhibitors from the haemo- the thrombin-fibrinogen interaction. These com- lymph of other have been implicated in pounds, could possibly affect exosite I on thrombin, the immune response of these organisms, including the specific fibrinogen active-binding site on throm- melanization processes, and haemolymph clot for- bin (see Ciprandi et al. 2006; Ricci et al. 2007; mation (Kanost, 1999). Furthermore, serine protease Koh and Kini, 2009; Tanaka-Azevedo et al. 2010). In inhibitors regulate digestive serine proteases (Morris the case of the CE, the thrombin inhibitor(s) and Sakanari, 1994; Azzolini et al. 2005), although attenuated hydrolysis of the peptide synthetic sub- in P. formica, digestion of the bloodmeal results strate by thrombin; they may have bound the primarily from cysteine protease activities (Manship catalytic site of thrombin (Horn et al. 2000), or to et al. 2008). Characterization of trypsin inhibitors different regions that prevented interaction of the from homogenates of haematophagous ectoparasites, catalytic site with the substrate (see Koh and Kini, such as ticks and the horn fly (see Azzolini et al. 2003, 2009; Tanaka-Azevedo et al. 2010). The thrombin 2005), have led to the identification of potential inhibitor(s) present in peaks P2 and P3, may function vaccine targets (see below). alongside anticoagulants present in P1 to inhibit clot Alongside these trypsin inhibitors, an anti- formation, and/or may reduce thrombin mediated coagulant(s) contained in the P1 fraction significantly thrombocyte aggregation to the site of lesion (see delayed recalcification time of human plasma. Mann and Lorand, 1993; Dahlbäck, 2000; Tavares- Heating of the P1 preparation destroyed this anti- Dias and Oliveira, 2009; Tanaka-Azevedo et al. coagulant activity, suggesting that the anticoagulant 2010). compound(s) present are proteinaceous; since P1 It is probable that the anticoagulant(s) identified in only slightly reduced thrombin interaction with the present study are contained in the salivary glands fibrinogen, we hypothesize that the anticoagulant(s) of this gnathiid, in a similar manner to parasitic inhibit a different factor(s) in the intrinsic pathway. cymothoid isopods (Romestand and Trilles, 1976a, However, the limited amount of gnathiid material b), or they may be divided between the salivary glands Blood feeding in Paragnathia formica 751 and digestive tract (anterior hindgut) as reported their fish hosts whilst feeding (see Smit and Davies, in Rhipicephalus microplus (syn, Boophilus microplus) 2004; Hayes et al. 2007, 2011). It is thus likely that as (Canestrini, 1888) ticks (Ricci et al. 2007) where they in other blood feeding ectoparasites, the isopod saliva delay clot formation during digestion in the midgut. contains a variety of immunomodulatory compounds In larger blood-feeding ectoparasites, for example (Singh and Girschick, 2003), such as prostaglandin mosquitoes, tabanid flies or ticks, similar biochemical E2 secreted by the salmon louse L. salmonis (see investigations have been conducted using proteins Wagner et al. 2008), alongside the potential trypsin exclusively extracted from the salivary glands inhibitors and anticoagulant(s) detected in this study. (Ribeiro et al. 1995; Stark and James, 1995, 1996; A fuller understanding of host-preference or Kazimírová et al. 2001, 2002), or proteins collected specificity is also vital in estimating the ecological after pilocarpine-induced salivation in ticks (Ciprandi and economic impact of gnathiid blood feeding. et al. 2006). However, the size of the gnathiid Molecular determination of host origin from the juveniles and in particular the unfed zuphea 1 stage dietary content of invertebrates has been reported for (1 mm each) (Upton, 1987), prevented dissection and predatory beetles (Zaidi et al. 1999), spiders (Agustí separation of the salivary glands (*100–200 μmin et al. 2003), blood-feeding ticks (Pichon et al. 2003), size) of P. formica, and therefore specific localization scavenging amphipod crustaceans (Blankenship and of the anticoagulant(s) could not be achieved. Yayanos, 2005), and recently the juveniles of gnathiid A recent report detailing the recovery of salivary isopods from the Great Barrier Reef (GBR) (Nagel secretions from the honeybee mite Varroa destructor and Lougheed, 2006; Jones et al. 2007). The latter (approximate size of 1–1·8 mm wide by 1·5–2mm authors amplified fish DNA from the bloodmeal long) after inducing salivation using pilocarpine contents of 2 nocturnal species of gnathiids from (Richards et al. 2011) could help future investigations the GBR, identifying 21 different host families, and into the localization and extraction of bioactive finding differences in host-preference between these compounds from gnathiid isopods. In this context, species. a comparative study of anticoagulants present in Results presented here, reinforce the relevance of unfed and fed stages of gnathiid juveniles, may reveal using molecular markers for identifying gnathiid host whether these bioactive compounds are restricted to a families and species, and it is surprising how many given stage, as previously reported for the cysteine- fish families/species were identified from relatively like proteolytic enzymes, which are more abundant few gnathiids inhabiting mud banks at Wells-next- in P. formica praniza stages (Manship et al. 2008). the-Sea and the Dovey estuary. It is generally as- Interestingly, experiments conducted on anticoagu- sumed that 18S genomic rDNA evolves more slowly lants of blood-feeding female mosquitoes revealed a than 16S mitochondrial rDNA, and is thus, highly strong factor Xa inhibitor in salivary gland extracts, conserved across a wide range of taxa (Pichon et al. whilst extracts of non-haematophagous male mos- 2003). This might explain our results, since some fish quitoes produced no delay in the recalcification species apparently shared a high level of 18S rDNA time of human plasma (Stark and James, 1995). sequence conservation, and gnathiid hosts could be A comparative study of anticoagulant compounds assigned only to their respective fish families. found in the haematophagous juvenile stages and Sequences obtained using the universal (vertebrate non-feeding males of P. formica (Monod, 1926) and invertebrate) 16S rDNA primers (Simon, 1991) might offer an insight into the function of the male were all of fish origin, corroborating findings by salivary glands. Blankenship and Yayanos (2005) that these primers Bioactive salivary compounds of arthropods of preferentially amplify vertebrate DNA. Importantly, medical, veterinary and economic importance have fish hosts were identified to the species level, by this been studied in detail (see Valenzuela, 2002; Ribeiro marker, making it ideal for studying gnathiid host- and Francischetti, 2003; Koh and Kini, 2009). A preferences as reported by Jones et al.(2007). rationale for characterizing these salivary proteins is In the present study, hosts of P. formica pranizae the design of and arthropod-borne control belonged to different fish families to those reported strategies, now advanced for anti-tick vaccines with for gnathiids on the GBR (Jones et al. 2007). Early successful applications in field studies (see Willadsen, laboratory feeding experiments using P. formica 2006). In aquatic environments, such studies have juveniles (Monod, 1926) and recent observations of been conducted only on the haematophagous marine gnathiids from the GBR (Nagel and Grutter, 2007), ectoparasite Lepeophtheirus salmonis Krøyer, 1837 suggest adaptability by these isopods to available fish (Copepoda: Caligidae). Prostaglandin E2 (an anti- hosts. In the case of Paragnathia, this monotypic inflammatory molecule) was identified in secretions genus has been reported, as noted earlier, across of the copepod salmon louse (see Wagner et al. 2008), Western Europe, North Africa and the Mediterranean and knockdown of the prostaglandin E synthase 2 basin (Monod, 1926; Upton, 1987; Menezes, 1984; gene was attempted (Campbell et al. 2009) to identify Cadée et al. 2001; Silva et al. 2006; Kirkim et al. targets for salmon vaccine development (Raynard 2008) where juveniles likely feed on a variety of fish et al. 2002). Gnathiid isopods cause tissue damage to hosts according to availability. The host families B. M. Manship and others 752 obtained here agree with the findings of Monod Agustí, N., Shayler, P., Harwood, J. D., Vaughan, I. P., Sunderland, K. D. and Symondson, W. O. C. (2003). Collembola as (1926), who noted that P. formica fed on members alternative prey sustaining spiders in arable ecosystems: prey detection of the Pleuronectiformes and Perciformes, and on within predators using molecular markers. Molecular Ecology 12, 3467– European eels. Severe pathological effects on eels 3475. doi: 10.1046/j.1365-294X.2003.02014.X. Andreotti, R., Gomes, A., Malavazi-Piza, K. C., Sasaki, S. D., have been reported as well as host death in heavy Sampaio, C. A. M. and Tanaka, A. S. (2002). BmTI antigens induce a infestations, or in captive environments (Mugridge bovine protective immune response against Boophilus microplus tick. and Stallybrass, 1983). Monod (1926) also observed International Immunopharmacology 2, 557–563. doi: 10.1016/S1567-5769 (01)00203-X. an accidental association between P. formica and Azzolini, S. S. A., Sasaki, S. D., Campos, I. T. N., Torquato, R. J. S., the European sprat, Sprattus sprattus (syn, Clupea Juliano, M. A. and Tanaka, A. S. (2005). The role of HiTI, a serine sprattus) (Linnaeus, 1758), a species sharing over protease inhibitor from Haematobia irritans irritans (Diptera: Muscidae) in the control of fly and bacterial proteases. Experimental Parasitology 111, 98% 16S rDNA homology with Clupea harengus 30–36. doi: 10.1016/j.exppara.2005.03.013. (Atlantic herring). However, Monod’s(1926) “acci- Azzolini, S. S. A., Sasaki, S. D., Torquato, R. J. S., Andreotti, R., dental association” may not be so, since C. harengus Andreotti, E. and Tanaka, A. S. (2003). Rhipicephalus sanguineus trypsin fi inhibitors present in the tick larvae: isolation, characterization, and partial DNA was ampli ed from 2 juvenile gnathiids at primary structure determination. Archives of Biochemistry and Biophysics Wells-next-the-Sea. Finally, unidentified gnathiid 417, 176–182. doi: 10.1016/S0003-9861(03)00344-8. isopods have recently been recorded on the European Blankenship, L. E. and Yayanos, A. A. (2005). Universal primers and fl ff PCR gut contents to study marine invertebrate diets. Molecular Ecology 14, ounder o the coast of Portugal (Cavaleiro and 891–899. doi: 10.1111/j.1365-294X.2005.02448.X. Santos, 2009), and this fish appears to be a common Cadée, G. C., Checa, A. G. and Rodriguez-Tovar, F. J. host for P. formica (see Menezes, 1984), as reported (2001). Burrows of Paragnathia (Crustacea: Isopoda) and Bledius (Arthropoda: Staphylinidae) enhance cliff erosion. International here. The success of the host typing technique could Journal for Plant and Traces 8, 255–260. doi: 10.1080/ lead to future identification of gnathiid hosts from 10420940109380193. larger specimen collections, to determine whether Campbell, E. M., Pert, C. C. and Bowman, A. S. (2009). RNA- fi interference methods for gene-knockdown in the sea louse, Lepeophtheirus they are host-speci c or respond to host availability. salmonis: studies on a putative prostaglandin E synthase. Parasitology 136, This is important since Marino et al.(2004) 867–874. doi: 10.1017/S0031182009990357. suggested that the opportunistic behaviour of juven- Cavaleiro, F. I. and Santos, M. J. (2009). Seasonality of metazoan ectoparasites in marine European flounder Platichthys flesus ile gnathiids could result in infestations of captive or (Teleostei: Pleuronectidae). Parasitology 136, 855–865. doi: 10.1017/ cultured fish worldwide, leading to economic loss. S003118200900626X. In conclusion, this research was undertaken to Ciprandi, A., De Olivera, S. K., Masuda, A., Horn, F. and Termignoni, C. (2006). Boophilus microplus: its saliva contains microphilin, provide important insight into the host-parasite a small thrombin inhibitor. Experimental Parasitology 114,40–46. doi: interactions that occur between the post-marsupial 10.1016/j.exppara.2006.02.010. stages of a gnathiid isopod and their host fishes. Dahlbäck, B. (2000). Blood coagulation. The Lancet 355, 1627–1632. doi: 10.1016/S0140-6736(00)02225-X. Together with our previous work on protein diges- Davies, A. J. and Smit, N. J. (2001). The life cycle of Haemogregarina tion in P. formica (see Manship et al. 2008), this bigemina (Adeleina: Haemogregarinidae) in South African hosts. Folia constitutes the first thorough investigation of blood Parasitologica 48, 169–177. Doolittle, R. F. (2011). Coagulation in vertebrates with a focus on evolution ingestion and host origin in P. formica juveniles, and inflammation. Journal of Innate Immunity 3,9–16. doi: 10.1159/ advancing considerably our understanding of feeding 000321005. in this extraordinary gnathiid isopod. Dreyer, H. and Wägele, J. W. (2001). Parasites of crustaceans (Isopoda: Bopyridae) evolved from fish parasites: molecular and morphological evidence. Zoology 103, 157–178. ACKNOWLEDGEMENTS Ferreira, M. L., Smit, N. J., Grutter, A. S. and Davies, A. J. (2009). A new species Gnathia aureamaculosa n. sp. (Crustacea. Isopoda. Gnathiidae) We are grateful to the Royal Society for the Protection parasitising teleosts from Lizard Island, Great Barrier Reef. Journal of of Birds, Ynyshir Nature Reserve, for allowing access to Parasitology 95, 1066–1075. doi: 10.1645/GE-1920.1. the Dovey Estuary. Many thanks to Drs Idir Akhouayri, Ferreira, M. L., Smit, N. J., Grutter, A. S. and Davies, A. J. (2010). Polly Hayes, Mike Johnston and Professor Nico Smit Gnathia grutterae sp. nov. (Crustacea: Isopoda) parasitising representatives of the teleost families Balistidae and Labridae from Lizard Island, Great for assisting with gnathiid collections. We are indebted to Barrier Reef, Australia. Zootaxa 2718,39–50. Drs Connor Jones and Laura Nagel for helpful advice on Hanspal, J. S., Bushell, G. R. and Ghosh, P. (1983). Detection of the sequencing of gnathiid material, and to the anonymous protease inhibitors using substrate-containing sodium dodecyl sulfate- reviewers for their valuable comments for improving this polyacrylamide gel electrophoresis. Analytical Biochemistry 132, 288–293. manuscript. doi: 10.1016/0003-2697(83)90010-6. Hayes, P. M., Smit, N. J. and Davies, A. J. (2007). Pathology associated with parasitic juvenile gnathiids feeding on the puffadder shyshark, FINANCIAL SUPPORT Haploblepharus edwardsii (Voight). Journal of Fish Diseases 30,55–58. doi: 10.1111/j.1365-2761.2007.00777.X. The studentship awarded to B. M. by the Biomedical and Hayes, P. M., Smit, N. J., Grutter, A. S. and Davies, A. J. (2011). Pharmaceutical Sciences Research Group, Kingston Unexpected response of a captive blackeye thicklip, Hemigymnus melapterus University, is gratefully acknowledged. (Bloch), from Lizard Island, Australia, exposed to juvenile isopods Gnathia aureamaculosa Ferreira & Smit. Journal of Fish Diseases 34, 563–566. doi: 10.1111/j.1365-2761.2011.01261.X. REFERENCES Horn, F., Coutinho dos Santos, P. and Termignoni, C. (2000). Boophilus microplus anticoagulant protein: an antithrombin inhibitor isolated from the Abebe, M., Ribeiro, J. M. C., Cupp, M. S. and Cupp, E. W. (1996). cattle tick saliva. Archives of Biochemistry and Biophysics 384,68–73. doi: Novel anticoagulant from salivary glands of Simulium vittatum 10.1006/abbi.2000.2076. (Diptera: Simuliidae) inhibits activity of coagulation factor V. 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